Note: Descriptions are shown in the official language in which they were submitted.
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USE OF OXALATE DEFICIENT ASPERGILLUS NIGER STRAINS FOR PRODUCING
A POLYPEPTIDE
Id of the invention
The invention relates to oxalate deficientAspergillus niger strains for
producing a
s polypeptide, to their use and to a method for obtaining such strains.
Background of the invention
Oxalic acid is an undesirable by-product that accumulates in the culture
supernatant of cells during fermentation and causes difficulties in the
downstream
~o processing of the desirable compound.
Four Russian prior art documents, named-Ru1-Ru4 (defined hereafter), describe
how to obtain oxalate deficient Aspergillus niger (A. niger) strains using
classical
mutagenesis methods. Oxalate deficient A. niger strains are defined as strains
that
produce less oxalic acid than the parental strain they originate from. They
demonstrate
15 that the choice of the mutagen agent is not critical: UV, or chemicals, or
a combination of
both as mutagen agents would lead to the obtention of oxalate deficient A.
niger strains.
They use a chromatography assay to select strains that produce less oxalic
acid or more
citric acid than the parental strain they originate from. They do not envisage
to use these
strains for producing polypeptides.
20 -Ru1: On methods of selecting A. niger mutants with altered capacity to
synthesize organic acids, ID Kasatkina and E.G. Zheltova, Mikrobiologiya, vol
34, no 3,
p511-518, May-June 1965.
-Ru2:RU2089615, New strains of A. niger has properties of producer of citric
acid
and can be used in microbiological industry (DW1998-249164).
25 -Ru3:The variability of A. niger, a producer of citric acid, under the
influence of
the separate and combined action of nitrosomethylurea and ultraviolet rays,
E.Y.
Shcherbakova, Z.S. Karadzhova and V.P. Ermakova, Mikrobiologiya, vol 43, no 3,
p508-
513, May-June 1974.
-Ru4: Change in the ratio of citric acid and oxalic acids in A. niger under
the
ao influence of mutagenic factors, V.M. Golubtsova, E.Y. Shcherbakova, L.Y.
Runkovskaya
and V.P. Eramkova, Mikrobiologiya, vol 48, no 6, p1060-1065, November-December
1979.
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2
Another publication, WO 00/50576 describes that oxaloacetate hydrolase
deficient host cells can be used for producing desirable compounds, such as
polypeptides, primary and secondary metabolites. These host cells have less
oxaloacetate hydrolase activity than the parental cells they originate from.
As a result,
these oxaloacetate hydrolase (OAH) deficient cells produce less oxalic acid
than the
parental cells they originate from. This patent application does not show
experimental
data demonstrating that an oxaloacetate hydrolase deficient cell is a suitable
polypeptide
producer. Furthermore, Pedersen et al, (Pedersen, H., et al, Metabolic Eng.,
(2000) 2,
34-41 ) later described that oxaloacetate hydrolase deficient Aspergillus
niger strains
1o transformed with a DNA construct comprising the DNA sequence encoding the
glucoamylase enzyme are not able to produce the glucoamylase enzyme at the
level the
wild type strain they originate from does under the same culture conditions:
the mutants
produce 50°l° less glucoamyiase than the wild type. Such a
mutant is not suited as a
polypeptide producer in an industrial setting.
There is still a need for oxalate deficient A. niger strains that are able to
produce at least
the amount of a polypeptide a wild type strain would produce and that can be
used as
polypeptide producer in an industrial setting.
2o Detailed description of the invention
Oxalate deficient A. niger strains suitable for the production of a given
polypeptide or
enzyme in an industrial setting have been isolated, wherein surprisingly the
oxalate
deficient strain produce at least the same amount of polypeptide or enzyme as
the wild
type strain they originate from under the same culture conditions. Preferably,
the mutants
produce at least the amount of polypeptide or enzyme the A, niger strain CBS
513.88
produces under the same culture condition.
In this application, A. niger strain CBS 513.88 is taken as a reference of
wild type oxalate
levels obtainable in an A. niger culture, as a reference of wild type
polypeptide level
ao obtainable in an A. niger culture and as a reference of intracellular OAH
activity obtainable
in an A, niger culture. Oxalate deficient A. niger strains are defined as
strains that produce
less oxalate than the A. niger strain CBS 513.88 under the same culture
conditions.
Preferably, the oxalate deficient A. niger strains used produce no more than
half the
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3
amount of oxalate that the wild type strain they originate from produces under
the same
culture conditions. More preferably, the oxalate deficient A. niger strains
used produce no
more than one third of the amount of oxalate that the wild type strain they
originate from
produces under the same culture conditions. Most preferably, the oxalate
deficientA. niger
s strains used produce no more than one fifth of the amount of oxalate that
the wild type
strain they originate from produces under the same culture conditions. More
preferably, the
oxalate deficient A. nigerstrains used produce no more than half the amount of
oxalate that
the A. niger strain CBS 513.88 produces under the same culture conditions.
More
preferably, the oxalate deficient A. nigerstrains used produce no more than
one third of the
~ o amount of oxalate that the A. niger strain CBS 513.88 produces under the
same culture
conditions. Most preferably, the oxalate deficient A. niger strains used
produce no more
than one fifth of the amount of oxalate that theA. niger strain CBS 513.88
produces under
the same culture conditions. According to a preferred embodiment of the
inventon, the
oxalate deficient A, niger strain used has been obtained by applying the
method defined
15 later in this application.
Preferably, the oxalate deficient A. niger strains of the invention are
strains that produce
more of a given polypeptide than the wid type strain they originate from under
the same
culture conditions. More preferably, the oxalate deficientA, nigerstrain
produces more of a
2o given polypeptide than the A. niger CBS 513.88 under the same culture
conditions.
A large variety of systems for detection of polypeptide are known to the
skilled person.
Detection systems include any possible assay for detection of polypeptide or
enzymatic
activity. By way of example these assay systems include but are not limited to
assays
25 based on colorimetric, photometric, turbidimetric, viscosimetric,
immunological, biological,
chromatographic, and other available assays.
Preferably, if the polypeptide produced is an enzyme, the amount of active
enzyme
produced is determined by measurement of its activity in a model reaction (see
examples).
Preferably, the oxalate deficient A. niger strains of the invention are
strains having a
detectable intracellular OAH activity as detected in a model reaction (see
experimental
information in the Examples) More preferably, the oxalate deficientA.
nigerstrains of the
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invention are strains having an intracellular OAH activity, which is ranged
between 0.1
and 100 % of the intracellular OAH activity of the wild type strain they
originate from as
detected in a model reaction, preferably between 0.5 and 90, more preferably
between
0.5 and 80, even more preferably between 1 and 50, most preferably between 1
and 25
and even most preferably between 1 and 10. According to another preferred
embodiment, the oxalate deficient A. niger strains have an intracellular OAH
activity,
which is ranged between 0.1 and 100% of the intracellular OAH activity of the
CBS
513.88 deposited strain as detected in a model reaction. More preferably, the
oxalate
deficient A. niger strains of the invention are strains having an
intracellular OAH activity,
~o which is ranged between 1 and 90% of the intracellular OAH activity of the
CBS 513.88
deposited strain as detected in a model reaction.
The existence of such oxalate deficient strains still having a detectable OAH
activity, is
surprising, since it was thought that OAH was the only molecule responsible
for the
formation of oxalate. Mutants still having detectable level of OAH activity
have several
~ s advantages compared to oxalate deficient strains with no detectable OAH
activity
(Pedersen H et al, Metabolic Eng. (2000) 2, 34-41 ): they are able to produce
at least the
amount of a given polypeptide the wild type strain would produce under the
same culture
conditions. Furthermore, the endogenous metabolic pathway of organic acids is
most
likely not pertubated.
zo According to a further preferred embodiment, the oxalate deficientA. niger
strain of the
invention is characterized by the fact that when this strain has been
transformed with an
expression construct comprising a gene coding for a polypeptide, said strain
produces at
least the amount of the polypeptide the wild type strain it originates from
would produce
under the same culture conditions, when the wild type strain has also been
transformed
25 with the same expression construct as the oxalate deficient strain.
The gene coding for the polypeptide to be produced may be homologous or
heterologous to the oxalate deficient A. niger strain used. The term
"heterologous"
means that the polypeptide is not native to the A. niger cell. Preferably, the
gene
so comprised in the expression construct is a heterologous gene for A. niger.
Preferred heterologous polypeptide is human serum albumine, lactoferrin,
chymosin or
Phospholipase A2. According to a preferred embodiment of the invention, the
oxalate
deficient strain has been transformed with a DNA construct comprising a DNA
sequence
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encoding said polypeptide. Preferably, the polypeptide is an enzyme. Enzymes
that can
be produced are carbohydrases, e.g. cellulases such as endoglucanases, (3-
glucanases,
cellobiohydrolases or ~3-glucosideases, hemicellulases or pectinolytic enzymes
such as
xylanases, xylosidases, mannanases, galactanases, galactosidase,
s rhamnogalacturonases, arabanases, galacturonases, lyases, or amylolytic
enzymes;
phosphatases such as phytases, esterases such as lipases, proteolytic enzymes,
oxidoreductases such as oxidases, transferases, or isomerases. Preferably, the
amylolytic enzyme to be produced is an alpha amylase (EC 3.2.1.1., alpha-1,4-
glucan-4-
glucano hydrolase or EC 3.2.1.2) . More, preferably, the DNA sequence encodes
a
~o fungal alpha amylase. Most preferably, the DNA sequence encoding the fungal
alpha
amylase is derived from A. niger or Aspergillus oryzae. According to another
embodiment, the enzyme to be produced is a proline specific endoprotease (EC
3.4.16.2). According to another embodiment, the enzyme to be produced is a
phospholipase A1 (PLA1 ) (EC 3.1.1.32). More, preferably, the DNA sequence
encodes a
fungal PLA1. Most preferably, the DNA sequence encoding the fungal PLA1 is
derived
from Aspergillus niger or Aspergillus oryzae.
The DNA sequence encoding the polypeptide to be produced may be operably
linked to
appropriate DNA regulatory regions to ensure a high level of expression of
said DNA
2o sequence and preferably a high secretion level of said polypeptide. If the
polypeptide to
be produced is native to Aspergillus niger, its native secretion signal is
preferably used.
Alternatively, if the polypeptide to be produced is not native toAspergillus
niger, a fusion
construct is preferably made comprising the glucoamylase gene of Aspergillus
niger
fused to the heterologous gene to be produced. According to a preferred
embodiment of
the invention, the regulatory regions of the Aspergillus oryzae alpha amylase
gene are
used. According to a more preferred embodiment of the invention, the
regulatory regions
of the A. niger glucoamylase gene are used. According to a preferred
embodiment of the
invention, the alpha amylase secretion signals are used. The DNA construct may
also
comprise a selectable marker. Alternatively, the selectable marker may be
present on a
so second DNA construct. By way of example these markers include but are not
limited to
amdS (acetamidase genes), auxotrophic marker genes such as argB, trpC, or pyre
and
antibiotic resistance genes providing resistance against e.g. phleomycin,
hygromycin B
or 6418. Preferably, the marker gene is the acetamidase gene from Aspergillus
nidulans.
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More preferably, the acetamidase gene from Aspergillus nidulans is fused to
the gpdA
promoter. Transformation methods of A. niger are well-known to the skilled
person
(Biotechnology of Filamentous fungi: Technology and Products. (1992) Reed
Publishing
(USA); Chapter 6: Transformation pages 113 to 156). The skilled person will
recognize
that successful transformation of A, niger is not limited to the use of
vectors, selection
marker systems, promoters and transformation protocols specifically
exemplified herein.
After transformation, typically, theA. niger population is cultivated on a
solid medium in a
petri dish. The transformants selected after culture on solid medium are
typically
cultivated in flask during three to seven days to check for expression of the
polypeptide.
Typically, for producing the polypeptide in the oxalate deficient A. niger
strain in an
industrial setting, a fed-batch fermentation process may be used. At the end
of the
fermentation, the polypeptide can be purified following techniques known to
the skilled
person. An example of such a recovery technique is explained in the following.
When
the fermentation is stopped, the host must be killed. This is accomplished by
adding a
killing-off agent at some specific temperature where this agent can work
effectively.
For example, the killing-ofF agent may be natriumbenzoate or kaliumsorbate.
Depending on the identity of the killing-off agent chosen, the broth
temperature is
adjusted to the corresponding working temperature of this agent, by using
classical
zo cooling methods known to the skilled person. In the case of a polypeptide
which is
secreted into the fermentation medium, the separation of the cell material
from the
polypeptide is for example a simple filtration process: the fermentation broth
is filtrated
using a membrane filter press equipped with a textile cloth (membrane filter
press and
textile cloth can be obtained from Harborlite). To improve the filtration
performance, a
2s suitable filter-aid can be used, together with a suitable pre-coat of the
filter cloth.
To remove any remaining small particles, additional filtration steps can be
carried out,
in such a way that a clear filtrate can be obtained. The filtrate can be
polished filtered
on filter plates with an average pore size of typically 1-10 micron. Several
types of
3o filter plates are 'known to the skilled person and are here suitable.
Subsequently, a
germ filtration may be carried out using a filter with a pore size of about
0.4
micrometer, to remove the major part of microorganisms. With these two
filtrations, a
pre-coat may be used to improve the filtration performance. The filtrate may
be then
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7
concentrated by ultrafiltration (UF) with a factor of typically 10-25. Several
types of OF
membranes are suitable here. During OF molecules with a typical molecular
weight of
less than a few thousands (depending also on the shape of the molecules) are
removed from the filtrate. Thus, the relative amount of low molecular weight
molecules
s to the polypeptide of interest may be reduced about 10-25 times after UF.
The
duration of the OF varies depending on the viscosity and filterability of the
filtrate
(which varies due to natural variations in the raw materials). At that stage,
the
concentration of the polypeptide present in the ultrafiltrate is usually high
enough to
proceed with the formulation of the polypeptide into either a liquid or a dry
formulation
~o depending on the application contemplated.
A method was developed for obtaining oxalate deficientA. niger strains which
are
suitable for producing high yields of a polypeptide and which can be used as
polypeptide
producers in an industrial setting. The polypeptide may be homologous or
heterologous
15 for said A, niger. In case of a heterologous polypeptide or enzyme, the
wild type strain on
which the method of the invention is applied may have been earlier transformed
to
express a gene coding for such polypeptide or enzyme as has been described
earlier in
the description. Such oxalate deficient A. niger strains produce at least the
amount of
polypeptide the wild type strains they originate from produce under the same
culture
Zo conditions. Preferably, the oxalate deficient A, niger strains produce more
polypeptide
than the wild type strain they originate from under the same culture
conditions. According
to another preferred embodiment, the mutants produce at least the amount of
polypeptide the A. niger strain CBS 513.88 produced under the same culture
condition.
More preferably, the mutants produce more polypeptide than the A. niger strain
CBS
25 513.88 produced under the same culture conditions.
This method comprises the following steps:
a) A. niger is subjected to UV irradiation,
b) MTP cultures of surviving colonies obtained in a) are realized
so c) a selection within the MTP cultures is performed in which mutants are
selected that produce no more than half the amount of oxalate that the wild
type strain they originate from produces under the same culture conditions,
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d) a second selection is performed within the mutants obtained in step c) in
which mutants are selected that produce at least the amount of polypeptide
the wild type strains they originate from produce under the same culture
conditions.
According to a preferred embodiment, the method comprises the following steps:
a) culture conditions are developed, which allow a production of at least 15
mM
oxalate in microtiterplates (MTP) or at least 30 mM oxalate in flask culture
in
the fermentation medium at the end of fermentation,
~o b) A. nigeris subjected to UV irradiation,
c) MTP cultures of surviving colonies obtained in b) are realized under the
culture conditions retained in a),
d) a selection within the MTP cultures is performed in which mutants are
selected that produce no more than half the amount of oxalate that the wild
type strain they originate from produces under the same culture conditions,
e) a second selection is performed within the mutants obtained in step d) in
which mutants are selected that produce at least the amount of polypeptide
the wild type strains they originate from produce under the same culture
conditions.
According to another preferred embodiment, the method comprises the following
steps:
a) culture conditions are developed, which allow a production of at least 15
mM
oxalate in microtiterplates (MTP) or at least 30 mM oxalate in flask culture
in
the fermentation medium at the end of fermentation,
b) A. niger conidiospores are subjected to UV irradiation,
c) MTP cultures of surviving colonies obtained in b) are realized under the
culture conditions retained in a),
d) a selection within the MTP cultures is performed in which mutants are
selected that produce no more than half the amount of oxalate that the wild
ao type strain they originate from produces under the same culture conditions,
e) a second selection is performed within the mutants obtained in step d) in
which mutants are selected that produce at least the amount of polypeptide
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9
the wild type strains they originate from produce under the same culture
conditions.
According to another preferred embodiment, the method comprises the following
steps:
a) A. niger is subjected to UV irradiation,
b) MTP cultures of surviving colonies obtained in a)aare realized,
c) a selection within the MTP cultures is performed in which mutants are
selected that produce at least the amount of polypeptide the wild type strains
1 o they originate from produce under the same culture conditions.
d) a second selection is performed within the mutants obtained in c) in which
mutants are selected that produce no more than half the amount of oxalate
that the wild type strain they originate from produces under the same culture
conditions,
Each step of these processes is characterized further below.
According to a preferred embodiment of the invention, in a first step,
colonies ofA, niger
are first cultivated in a medium which allows a production of at least 30 mM
oxalate in
MTP or at least 100 mM oxalate in flask culture in the fermentation medium at
the end of
2o fermentation. The fermentation time should be at least 3 days. It is
further a preferred
embodiment of the method that the pH of this medium does not need to be
manually
corrected. The pH of the medium of this step is maintained between 3 and 7,
preferably
between 3,5 and 6,5, more preferably between 4 and 6. Most preferably the pH
of this
medium is maintained between pH 5 and 6. At such a pH value, the production of
oxalate
z5 is known to be high. The pH of the medium is preferably buffered with a
solution of 2-[N-
Morpholino~ethanesulfonic acid (MES) whose concentration is ranged between 0,1
and 1
M, more preferably between 0,15 and 0,55 M. Most preferably the MES
concentration is
0,5 M. A nitrogen source is present in the medium of this step. Preferably the
nitrogen
source is a nitrogen source, which does not result in the acidification of the
fermentation
so medium as a result of its uptake by the cell. More preferably, the nitrogen
source of the
medium of this step is urea. According to a preferred embodiment of the
present
invention, the medium used in this step is the flask defined medium 2 (FDM2)
(see
example 1 ). According to a preferred embodiment of the present invention, the
A, niger
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strain used in this step is WT2 or the A, niger strain CBS 513.88 (see
experimental
information).
In a second step, A. niger is subjected to UV irradiation so that the survival
percentage
5 is ranged between 0,01 % and 60%. Preferably, the survival percentage is
ranged
between 0,05% and 50%. More preferably, the survival percentage is 0,1%. It is
well
known to the skilled person that conidiospores is the preferred material to
mutagenizeA.
niger by physical or chemical means. Mutants may however also be obtained from
mycelium cells. The selection method described herein may be applied to select
mutants
~o obtained from either conidiospores or mycelium cells.
In a third step, MTP cultures of the surviving population obtained in a second
step is
performed during at least 3 days.
At the end of the MTP culture of the third step, mutants can be selected in a
fourth step on
basis of their oxalate production (oxalate selection step). Preferably mutants
are selected
that produce no more than one third of the amount of oxalate that the wild
type strain they
originate from produces under the same culture conditions. More preferably,
mutants are
selected that produce no more than one fifth of the amount of oxalate that the
wild type
2o strain they originate from produces under the same culture conditions.
An assay to quantify the oxalate present in the medium that may be used is
described in
the Examples. For practical reasons, the best mutants (the lowest oxalate
producers) are
retained for further characterization. Preferably 5 to 50 mutants are retained
for further
characterization. Typically, after 7 days in flask cultivation, it can be
checked that these
selected mutants produce far less oxalate than the wild type strain: in the
case described
in figure 7, less than 5 mM oxalate is found in the fermentation medium of the
mutants
compared to 40-45 mM for the wild type strain. After 7 days of fermentation,
it can further
be checked whether the medium is less acidified by the selected mutants than
by the
wild type strain. It can also be checked by measurement of the biomass
produced and by
ao measurement of the residual glucose concentration at different intervals
during
fermentation that the low level of oxalate measured in the mutants is not the
consequence of either a poor growth andlor a poor metabolic activity of the
selected
mutants.
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A second selection step which can be applied to the mutants before or after
the oxalate
selection step is the following: select mutants that produce at least the
amount of
polypeptide the wild type strains they originate from produce ur~ier the same
culture
s conditions. Preferably, the mutants produce more of a given polypeptide than
the wild type
strains they originate from under the same culture conditions. According to
another
preferred embodiment, the mutants produce at least the amount of a given
polypeptide the
A. niger strain CBS 513.88 produced under the same culture condition. More
preferably,
the mutants produce more of a given polypeptide than the A. niger strain CBS
513.88
~ o under the same culture conditions. To perform this last step, the mutants
obtained in the
previous step and a wild type control are cultivated in liquid medium for at
least three days
in a suitable medium. Preferably, the cultivation is performed during at least
five days. At
the end of the culture, the amount of the polypeptide produced may be
determined using a
system for detection of said polypeptide as defined earlier on in the
application. Preferably,
~ s if the polypeptide produced is an enzyme, the amount of active enzyme
produced is
determined by measurement of its activity in a model reaction (see examples).
An optional sixth step may be further applied to select for oxalate deficient
A. niger
strains having an intracellular OAH activity which is detectable as detected
in a model
2o reaction. Preferably, the model reaction is the one described in
experimental information
in the Examples. More preferably, this step allows the selection of oxalate
deficientA.
niger strains having an intracellular OAH activity, which is ranged between
0.1 and 100
of the intracellular OAH activity of the wild type strain they originate from
as detected
in a model reaction, preferably between 0.5 and 90, more preferably between
0.5 and 80,
25 even more preferably between 1 and 50, most preferably between 1 and 25 and
even
most preferably between 1 and 10. According to another preferred embodiment,
the
oxalate deficient A. niger strains have an intracellular OAH activity, which
is ranged
between 0.1 and 100% of the intracellular OAH activity of the CBS 513.88
deposited
strain as detected in a model reaction. More preferably, the oxalate
deficientA. niger
ao strains of the invention are strains having an intracellular OAH activity,
which is ranged
between 1 and 90% of the intracellular OAH activity of the CBS 513.88
deposited strain
as detected in a model reaction.
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The invention also relates to the use of an oxalate deficient A. niger strain
for
producing a given polypeptide. Accordingly, the invention also relates to a
method for
producing a given polypeptide wherein an oxalate deficient A, niger as defined
in this
application is used. Such strain produces at least the same amount of said
polypeptide
s as the wild type strain it originates from under 'the same culture
conditions. Preferably,
the strain produces more of said polypeptide than the wild type it originates
from
under the same culture conditions. According to another preferred embodiment,
the
strain produces at least the same amount of said polypeptide or enzyme as the
CBS
513.88 A. niger strain under the same culture conditions. More preferably, the
strain
~o produces more of said polypeptide or enzyme than the CBS 513.88 A. niger
strain
under the same culture conditions.
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Brief descrilption of the drawings:
Figure 1 depicts the oxalate assay standard curve. The measured optical
density
is given as a function of the oxalate concentration present in solution.
Figure 2 depicts the evolution of the pH of the culture supernatant of wild
type A.
niger during fermentation in FDM1 medium with or without pH correction.
Figure 3 depicts the average oxalate production obtained during fermentation
of
the wild type A. niger in the FDM1 medium with or without pH correction.
Figure 4 depicts the average oxalate production obtained during fermentation
of
~o the wild type A. nigerin the FDM1 medium as a function of the MES
concentration, with
ammonium or urea as nitrogen source, without pH correction.
Figure 5 depicts the average oxalate production obtained during fermentation
of
the wild type A. niger in the FDM2 medium without pH correction.
Figure 6 depicts the pH evolution during fermentation of wild type and some
selected oxalate deficient A. niger in the MDM1 medium.
Figure 7 depicts the average alpha amylase produced after fermentation in the
FDM2 medium by the wild type and the 34 mutants as a function of their oxalate
production.
Figure 3 depicts the measured OAH activity in three oxalate deficient A, niger
zo mutants and in the wild type.
Figure 9 depicts the average oxalate production obtained during the
fermentation
of the wild type and oxalate deficient A. niger in the FDM2 medium without pH
correction.
Figure 10 depicts the residual glucose concentration present during
fermentation
of wild type and oxalate deficient A. niger in the FDM 2 medium.
25 Figure 11 depicts the pH evolution of culture supernatants of wild type and
oxalate deficient A, nigerfermented in the FDM2 medium.
Figure 12 depicts the evolution of the biomass produced during fermentation of
the wild type and oxalate deficient A, niger in the FDM2 medium.
Figure 13 depicts the production of a proline specific endoprotease in WT1 and
in
ao FINAL (mutant 22) comprising the same estimated copy numbers of the gene
coding for
the proline specific endoprotease.
Figure 14 depicts the production of phospholipase A1 in WT1 and in FINAL
(mutant 22) in shake flask.
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14
EXAMPLES
Experimental information
Strains
WT 1: A, niger strain is used as a control for the level of oxalate,. the
level of a given
polypeptide and the level of intracellular OAH activity. This strain is
deposited at the CBS
Institute under the deposit number CBS 513.88.
~ o WT 2: WT 1 strain comprising several copies of an expression cassette
comprising the A. oryzae alpha-amylase gene integrated in the genome. This
gene was
already described elsewhere (Wirsel et al., (1989), Mol. Microbiol. 3:3-
14).The original
signal sequence coded by the A. oryzae alpha-amylase gene was replaced by the
one of
the glucoamylase gene from A. niger. WT 2 was constructed and selected by
techniques
known to persons skilled in the art and described in EP 635 574 A1 and in WO
98146772.
OAH activity assay
Shake flask fermentations of different A, niger strains were performed as
decribed
hereafter. Cells were cultivated at 30°C, 170 rpm for three days in
100m1 of OAH
2o cultivation medium in 500 ml shake flasks without a baffle. The OAH medium
is
defined in Table 1 below.Then, the pH was shifted to 8 by addition of Na2C03
and
cells were cultivated for an additional 15 to 18 hours. Mycelium was harvested
by
filtration, washed with 0.9°I° (w/v) NaCI, frozen in liquid
nitrogen and stored at
-80°C. Frozen cells were disrupted in a mortar under liquid nitrogen
and then
suspended in the following extraction buffer: 100 mM MOPS buffer pH 7.5 (MOPS
=
Morpholino propanesulfonic acid), 2 mM MnCl2, 20 mM DTT, 5% sucrose. The
suspension was centrifuged for 20 min. at 14,000 r.p.m. at 4°C in an
Eppendorf
centrifuge 54178. 925 p,l of the assay buffer (assay buffer: 100 mM MOPS pH
7.5 l 2
mM Mn 2+) was pre-heated at 25°C. 25 wl of a 40 mM oxaloacetic solution
was added
so to this preheated mix. The oxaloacetic solution was prepared by dissolving
0.053 g of
oxaloacetic acid in 10 ml of the assay buffer. 5,0 p,l of the suspension
obtained after
centrifugation was added to the preheated mix. OAH activity was determined
according to the method described by Pedersen et al, 2000, Mol. Gen. Genet.
263:281-286. Briefly, oxaloacetate is used as substrate. The enzyme activity
was
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determined from the rate of decrease of the absorbance (delta Almin) at 255 nm
during 3 minutes with a time interval of 20 seconds and the absorption
coefficient of
oxaloacetate. The assay was carried out at 25°C.
5 Table 1:
OAH medium
Trace Metal Solution
~o ZnS0~~7H20 0.143 g
CuS04~5H20 0.025 g
NiC12~6H20 0.005 g
FeS04~7H20 0.138 g
MnC12~4H20 0.060 g
15 Water up to 10 ml
OAH medium, pH = 2.5 or 4.5,
Sucrose 20 g
2o KHzP04 1.5 g
MgS04~7H20 1 g
NaCI 1 g
CaC12~2H~0 0.1 g
NaN03 15 g
Trace Metal solution
0.5 ml
(Adjust pH to 2.5
with HCI)
Water up to 1 liter
ao Protein assay
The protein content in the samples was determined according the Coomassie Plus
Protein assay with Bovine Serum Albumin as a standard according to the
manufacturer's instructions (Pierce, product number 23236).
In Example 1, alpha amylase is given as an example of enzyme that can be
produced by
an oxalate deficient A. niger strain at a level which is at least the same as
the one
produced by the parental strain the mutant originate from under the same
culture
conditions.
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16
Example 1
Method to make Oxalate deficient Asperg~illus niger mutants which are high
polypeptide producers
s Oxalate deficient A. niger mutants were made starting from WT2.
1. Growth media
Cultures were performed at 34°C, in 96-wells microtiter plates (MTPs)
or 300 ml
flasks with one baffle in a rotary shaker at a shaking speed of 220 rpm.
~o Flask precultures were inoculated with 17 000 spores per ml. 100 ml
cultures
were inoculated with 10 ml of preculture.
Table 2
Flask preculture medium 1 (FPM1), pH 5.5
15 (all components are given in grams per liter)
Corn steep 20
liquor
(Roquette-Freres,
France)
Glucose.l HaO 22
Tahla
Flask defined medium 1 (FDM1), pH 6
20 (all components are given in grams per liter)
Glucose.1 H20 82.5
Maldex 15 25
(Boom Mepel,
Netherlands)
Citric acid 2
NaH~P04.1 HBO 4.5
KH2P04 9
(NH4)2S04 15
ZnCl2 0.02
MnS04.1 HBO 0.1
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17
CuS04.5H20 0.015
CoC12.6H20 0.015
MgS04.7H20 1
CaC12.2H20 0.1
FeS04.7Hz0 0.3
MES* 30
(*2-[N-Morpholino]ethanesulfonic acid)
Flask defined medium 2 (FDM2), pH 6: the FDM2 medium had the same composition
as FDM1 except that 15 grams per liter urea are present instead of 15 grams
per liter
(NH4)ZS04. This medium contained 100 grams per liter MES instead of 30 grams.
Table 4
Microtiter plate defined medium 1 (MDM1), pH 6
(all components are given in grams per liter)
Glucose.1 H20 15
Citric acid 2
NaHZP04.1 HBO 1.5
KH2P04 3
Urea 5
ZnCl2 0.02
MnS04.1 Hz0 0.1
CuS04.5H2O 0.015
CoC12.6H20 0.015 ,
MgS04.7H20 1
CaC12.2H2O 0.1
FeS04.7H20 0.3
MES* 30
~o (*2-[N-Morpholino]ethanesulfonic acid)
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18
2. Assay for oxalate detection inA. nigerculture supernatant
A commercial kit available from Sigma diagnostics (Sigma. OXALATE diagnostic
s kit, catalogus. nr. 591 year 2000-2001 ) was employed for oxalate
quantification. The
volumes recommended by the manufacturer were downscaled to reach a final assay
volume of 48 ~I, the assay being performed in 384-wells MTPs. A Beckman
Multimek 96
was employed for all liquid transfers and the absorbance was read at 550 nm in
a BMG
spectrofluorimeter. The Oxalate assay standard curve is given in Figure 1 (the
optical
~ o density, OD, as a function of the oxalate concentration). In these
conditions, he assay was
found to be linear up to 2.5 mM.
3. Development of cultivation conditions to maximize oxalate production
The wild-type strain employed throughout this section is WT 1.
15 The pH has been described as the most critical parameter for oxalate
production. To
achieve a high oxalate production, the pH of A. niger cultures should be
maintained at a
value close to 6 (Ffubicek, C. P., et al, Appl. Environ. Microbiol. (1988) 54
, 633-637; and
Ruijter, G. J. G., et al,. Microbiology (1999) 145, 2569-2576). Oxalate
production sharply
decreases for pH values below 4 (Ruijter, G. J. G., van de Vondervoort, P. J.
I., and Visser,
2o J. 1999. Microbiology 145, 2569-2576). A pH close to 6 can hardly be
maintained in A.
niger cultures, because of the production of se~reral organic acids by the
fungus. To test
how critical the pH of the culture was in the FDM1 medium, triplicate flasks
cultures were
performed with a wild-type A, niger strain, either with or without daily
manual pH correction
by addition of sterile sodium hydroxyde. A pre-culture phase of 48 hours in
FPM1 medium
Zs was performed before FDM1 medium inoculation. In FDM1 medium, 0.15 M MES
(30 g iL)
was present to buffer the medium acidification during A. nigergrowth.
As can be seen in figure 2, the buffer p-esent in the medium was not
sufficient to
counterbalance the production of organic acids by A. niger, and figure 3 shows
that the
oxalate yield was greatly affected by the pH of the culture. Cultures in which
the pH was
so corrected yielded about 5 times more oxalate than the cultures in which the
pH was not
corrected.
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19
During the screening for oxalate deficient strains, A. niger was grown in
conditions
yielding a maximal oxalate production, so that oxalate deficient strains could
be selected
and easily distinguished from a strain producing wild-type levels of oxalate.
For practical
reasons, a manual pH correction could not be an option to achieve a maximal
oxalate
s production in the initial screening phase, when a huge number of mutants
were still under
evaluation. To improve the level of oxalate production without having the need
to correct
the pH of the cultures, two parameters were tuned in the FDM1 medium, which
were the
MES concentration in the medium and the nature of the nitrogen source.
As shown in figure 4, increasing the MES concentration and replacing ammonium
~ o sulfate by urea had a major impact on the maximal oxalate concentration,
which could be
reached in A. niger cultures without pH correction. In figure 5, the maximal
oxalate
concentration reached after 6 or 7 days of fermentation depending on the
composition of
the fermentation medium is represented.
1 M MES affected the growth ofA. niger and an intermediate concentration of
0.5 M
15 MES was chosen. Thus the growth medium finally chosen for flask cultivation
during the
screening was the FDM1 were the MES concentration was 0.5 M and where the
ammonium sulfate was replaced by urea. From now on, that medium will be
referred to as
FDM2.
Figure 5 shows that in FDM2, the oxalate concentration reached wthout pH
2o correction was equivalent to the oxalate concentration reached in FDM1 with
pH correction
(compare with figure 3). So, there was no need for pH correction anymore.
4. First selection: oxalate production
A. niger conidiospores were collected from WT 2 colonies sporulating on potato
25 dextrose agar (PDA) medium (Difco, POTATO DEXTROSE AGAR, cultivation
medium,
catalogus. nr. 213400, year 1996-1997). 10 ml of a suspension containing 4x1
conidiospores per ml was subjected to UV irradiation at 254 nm (Sylvania, 15
Watts Black
Light Blue tube, model FT15T8/BLB) until an energy of 0,1783 J/crr~ was
received. A
survival of 0.1 % of the initial number of colonies was obtained. The
mutagenized spores
so solution was plated on PDA medium and 10 000 survivors were picked using a
Genomic
Solutions Flexys colony picker and further grown into 96 wells microtiter
plates (MTP),
These MTPs, called "masterplates" were incubated at 34°C until a strong
sporulation was
apparent.
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The masterplates were replica plated using the Genomic Solutions Flexys colony
picker into MTPs containing 40 ~,I of FPM1 and incubated for 48 hours at
34°C. 170 ~,I of
MDM1 was then added and the MTPs were further incubated for 7 days at
34°C.
The supernatant of the 10 000 individual cultures was assayed for the presence
5 of oxalate. In the cultivation conditions employed, the oxalate
concentration reached in
cultures of the WT 1 and WT 2 strains was in the range of 40 mM. The mutants
for which
the oxalate concentration in the growth medium was below 12 mM were selected
for a
further selection round. 255 mutants were retained. This second selection
round was
more stringent than the first one, so that it allowed to get rid of false
positives.
~o The second mutant selection consisted of a quadruplicate MTP cultivation
and
assay for oxalate. The conditions employed were the same as the ones described
here
above. Table 5, second column below lists the oxalate concentration reached in
the
lowest producers amongst the mutants and in wild-type MTP cultures.
15 Table 5
Mutants
Average oxalateAverage alpha
concentrationamylase activity
(mM) (U/ml)
1 1.51 3.5
2 6.34 3.7
3 10.61 6.3
4 13.25 6.9
5 4.46 5.2
6 9.18 6.7
7 10.41 4.7
8 11.47 3.9
9 2.09 3.4
10 3.23 4.3
11 4.05 5.9
12 5.87 3.1
13 7.36 4.4
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21
14 9.82 3.3
15 2.5 7
16 1.28 3.1
17 2.86 4.6
18 2.39 5,1
19 5.71 5.8
20 4.19 4.5
21 2.25 6
22 0.78 5.4
23 0.5 3.9
24 1.38 4.3
25 6.42 6.6
26 7.16 5,2
27 2.28 3.9
28 2.33 4.5
29 8.15 5.5
30 3.21 5.3
31 3.7 4,6
32 1.84 4.4
33 1.87 4.8
34 8.54 4.4
WT 1 33.80 -
WT 2 36.80 1,7
1 U/ml is the quantity of alpha amylase needed to convert 1 g soluble starch
per hour
into a product. The formation of this product is being measured by following
the
absorption at 620 nm after addition of lode at pH 5.5 and at 30° C. The
incubation time
with iode is between 15 and 25 minutes.
Figure 6 shows that the selected mutant strains acidify less the MDM1 growth
medium upon growth compared to the wild-type strains.
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5. Second selection: alpha amylase production
As a second selection step, the 34 mutants obtained in the fiormer paragraph
were
subsequently selected as to their capacities to produce alpha amylase.
The 34 mutants and WT2 were grown the same way as in the former paragraph, and
characterized as to their alpha-amylase production.
The alpha-amylase activity present in culture supernatants was determined
using the
alpha amylase assay kit from Megazyme (Megazyme, CERALPHA alpha amylase assay
kit, catalogus. ref. K-CERA, year 2000-2001 ). Table 5 third column lists the
average
alpha amylase production detected in WT2 and in the 34 mutants.
~o Figure 7 depicts the average production of alpha amylase as a function of
the oxalate
production of the 34 mutants and the wild type. It could be observed in table
5, third
column and in figure 7 that all the 34 mutants produced significantly more
alpha-amylase
than the wild-type strain they originated from. All the oxalate mutants found
at the former
paragraph were retained as mutants able to produce at least the same amount of
enzyme as the wild type they originate from under the same culture conditions.
Mutants 15, 19 and 22 were selected for further selection.
6. Third selection: OAH activity
As an additional selection, the intracellular OAH activity was measured in the
zo three mutants (15, 19, 22) selected at the former paragraph and as a
control in WT1 and
WT2. For some strains, measurements were made twice (A, B) as indicated in
figure 8.
The test developed to measure OAH activity is described in experimental data.
Mutants
and 22 showed a detectable OAH activity (figure 8): approximatively 10 to 20 %
of the
WT 1 or WT2. Surprisingly mutant 19 showed a high OAH activity, which is
similar to the
zs one of WT2. Surprisingly, these three oxalate deficient mutants still have
a relative high
OAH activity. Furthermore, they also have good enzyme production capacities.
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23
Example 2
Characterisation of the A, niger oxalate deficient mutants
1. Growth media
Table 6
FPM1 and FDM2 media; as defined in example 1.
Flask preculture medium 2 (FPM2), pH 5.5
~o (all components are given in grams per liter)
Maltose.1 H20 30
Casein hydrolysate10
Yeast extract 5
KH2P04 1
Tween 80 3
MgS04.7H~0 0.5
ZnCl2 0.03
CaCl2 0.02
MnS04 0.01
FeS04.7H20 0.3
2. Characterization of the A, niger oxalate deficient mutants
Mutants 18, 22, 15, 23, 19, 33 were grown in the FDM2 medium, after 48 hours
of
preculture phase in FPM1, and characterized as to their oxalate production,
and several
growth parameters (residual glucose, pH and biomass formed).The results
obtained with
the FDM2 medium confirmed the low level of oxalate production of the mutants
compared to the wild-type strains (figure 9).
The residual glucose present in the FDM2 medium during growth of wild type and
2o mutant strains was assayed using the Glucose assay kit from Sigma
Diagnostics (Sigma,
GLUCOSE diagnostic kit, catalogus nr. 510-A, year 2000-2001 ). As can be seen
in figure
10, the glucose was almost completely consumed in some mutant cultures after 7
days
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24
of growth, suggesting the low oxalate level found in the selected mutant did
not reflect a
low metabolic activity. Only mutant 23 seemed to have a reduced metabolic
activity.
The pH of the cultures was also followed. As previously observed (see example
1 ), the acidification of the culture medium was less advanced in the mutants
than in wild-
s type cultures (See figure 11 ).
Finally, to ensure that the reduced oxalate production of the mutants was not
due
to a poor growth, the biomass formation was followed by weighing the biomass
dry
weight formed in the cultures at various cultivation times. Flasks were
sacrificed at each
time interval considered and the total biomass dry weight content of the flask
was
~o determined.
As can be seen in figure 12, the mutants showed various growth profiles but
tended to reach the same biomass level as the parental strain WT 2 after 7
days of
cultivation. Mutant 23 was the only one which shown a low level of biomass
formation,
but this level was still comparable to the one reached by the wild-type strain
WT 1 from
~ s which WT 2 originated. Mutant 23 was not retained as oxalate deficient
mutant for further
characterization. The sporulation capacities of the mutants were visually
evaluated. it
was found that the sporulation level of the mutants was comparable to the one
of the wild
type strain they originate from. Only one mutant seemed to have lower
sporulation
capacities.
2o In the following examples, mutant 22 was used as oxalate deficient A, niger
strain for
producing different enzymes. This mutant was obtained from WT2 and earlier on
from
WT1. In order to express other enzymes in this mutant, all the copies of the
alpha
amylase gene were deleted according to the method described in EP 635 574 A,
using
the acetamidase gene as selection marker gene. This mutant empty of any
foreign
25 enzyme encoding gene would be named FINAL in the following examples.
Subsequently,
FINAL was transformed with expression construct comprising the gene coding for
the
corresponding enzyme to be expressed as described in the following examples.
In order
to express specific enzymes in WT1, the expression constructs introduced in
FINAL
were also introduced in WT1 as described in the following examples. Copy
number was
so checked. Mutant 22 was tested and compared to WT1 for the production of a
proline
specific endoprotease and PLA1. Mutant 22 produced the same amount of all
enzymes
tested as the WT1 it originates from under the same culture conditions or even
more.
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Example 3
Comparison of the production of a proline specific endoprotease in the WT1
and in FINAL strains
5 The gene coding for the proline specific endoprotease, which has been used
has
already been published elsewhere (WO 02/45524). In order to express the
proline
specific endoprotease described in WO 02145524 in WT1 and in FINAL, the
construct
depicted in WO 02/45524 (pGBFIN11-EPO) was introduced in these strains by
cotransformation as described in WO 02/45524.
~ o Transformants with similar estimated copy number were selected to perform
shake
flask experiments in 100 ml of the medium as described in EP 635 574 A1 at
34°C
and 170 rpm in an incubator shaker using a 500 ml baffeled shake flask. After
four
days of fermentation, samples were taken to determine the proline specific
endoprotease activity. The proteolytic activity of the proline specific
endoprotease was
spectrophotometrically measured in time at pH 5 and about 37°C using Z-
Gly(cine)-
Pro(line)-pNA as a substrate. 1 U proline specific endoprotease is defined as
the
amount of enzyme which converts 1 micromol Z-Gly(cine)-Pro(line)-pNA per min
at
pH 5 and at 37°C.
2o Figure 13 shows that the proline specific endoprotease activity of the A.
niger
transformants with different estimated copy number is comprised in a range
from 42 to
135 U/I. Strains with one estimated copy number have an activity of 42- 46 U/I
and
correlates well with the activity of two and three copy strains. We concluded
that
FINAL produces at least the same amount of proline specific endoprotease as
WT1
25 under the same culture conditions.
Example 4
Comparison of phospholipase A1 (PLA1) production in WT1 and in FINAL
strains
ao We chose to express PLA1 from A. oryzae in WT 1 and in FINAL. The gene
encoding
this enzyme has already been published (Watanabe I, et al, Biosci. Biotechnol.
Biochem. (1999), Vol 63, numero 5, pages 820-826). This gene was cloned into
pGBFIN11 using the same technique as described in WO 02/045524 for the cloning
of
the proline specific endoprotease gene in pGBFIN11-EPO. This construct was
introduced in these strains by cotransformation as described in WO 02/45524.
Three
independent transformants of WT1 and FINAL were tested for PLA1 expression in
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26
shakeflasks. The transformants with similar estimated copy number were
cultivated in
100 ml of the same medium as described in EP 635 574 A1 at 34°C and 170
rpm in
an incubator shaker using a 500 ml baffeled shake flask. After 2, 3, 4, 5 days
of
fermentation, samples were taken to determine the PLA1 activity. To determine
s phospholipase PLA1 activity from Aspergillus niger (PLA1 )
spectrophotometrically, an
artificial substrate is used: 1,2-dithiodioctanoyl phophatidylcholine (diCB,
substrate).
PLA1 hydrolyses the sulphide bond at the A1 position, dissociating thio-
octanoic acid.
Thio-octanoic acid reacts with 4,4 dithiopyridine (color reagent, 4-DTDP),
forming4-
thiopyridone. 4-Thiopyridone is in tautomeric equilibrium with 4-
mercaptopyridine,
~ o which absorbs radiation having a wavelength of 334 nm. The extinction
change at that
wavelength is measured. One unit is the amount of enzyme that liberates of 1
nmol
thio-octanoic acid from 1,2-dithiodioctanoyl phosphatidylcholine per minute at
37°C
and pH 4Ø
15 The substrate solution is prepared by dissolving 1 g diC8 crystals per 66
ml ethanol and
add 264 ml acetate buffer. The acetate buffer comprises 0.1 M Acetate buffer
pH 3.85
containing 0.2% Triton-X100. The colour reagent is a 11 mM 4,4-
dithiodipyridine solution.
It was prepared by weighting 5,0 mg 4,4-dithiodipyridine in a 2 ml eppendorf
sample cup
and dissolving in 1.00 ml ethanol. 1.00 ml of milli-Q water was added. The
results are
2o depicted in figure 14. It is shown that PLA1 activity in transformants of
WT1 cultures
decreased after 4-5 days. However, the PLA1 activity of transformants of FINAL
accumulates during fermentation and no decrease in activity could be observed.
We
concluded that FINAL produces more PLA1 than the wild type counterpart it
originates
from under the same culture conditions.
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27
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to the microorganism referred to
in the description
on page 2 line 26 (and other pages)
B. H)ENTIFICATION OF DEPOSIT Further
deposits are identified on an additional
sheet
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CEN'fRA.Aa., B1J ZIAU VOOR SGIIIMMEI~CIJI;fURFS
Address of depositary institution
(including postal code and country)
Oosterstraat 1
P.O.Box 273
3740 AG BAARN
The Netherlands
Date of deposit 10 August 1988 Accession Number CBS 513.88
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